See also CRISP gene editing, RNA interference and Gene Delivery. 

Companies: Dark Horse Consulting Group

Gene therapy encompasses the genetic modulation of cells and/or tissue in order to achieve a therapeutic effect. Gene therapy comes in a variety of forms from standard gene replacement strategies (for loss of function abnormalities), to gene silencing strategies (for gain of function abnormalities) and gene editing techniques. In all cases, the therapeutic genetic material is packaged in either a recombinant virus or non-viral vehicle and is delivered to cells via either in vivo or ex vivo routes of admistration. (Henckaerts, “What re the issues associated with developing gene therapies for rare disease and are the current development models working?” Cell & Gene Therapy Insights 2024: 10(5), 773-784)

Cell and gene therapy (CGT) products represent a diverse class of advanced therapies with the potential to treat and cure the underlying cause of a disease. Over the last several years the number of FDA approved CGTs has steadily increased form two products in 2015 to a total of 28 in 2024 (excluding cord blood dervived therapies, off-market and withdrawn licenses). These 38 approvals are spread across 3 major product classes: cell therapies, gene therapies and gene-modified cell therapies. The majority of approved products utilize viral vectors to deliver genetic material, with adeno-associated virus (AAV) being the predominant platform for in vivo gene therapies and lentivirus (LVV) being the predominant modality sued ex vivo for gene modified cell therapies. AAV delivers genetic material that forms episomal DNA in a cell which largely does not integrate into the host genome. This leads to stable expression in non-dividing cells and transient expression in dividing cells, as each cell division will ultimately dilute the episomes. In contrast, LVV delivers its genetic payload into both dividing and non-dividing cells leading to stable expression, and integration is typically random. (Christina Fuentes “Coming of age: an overview of the growing toolbox for gene editing and its use in CGT applications” Cell & Gene therapy insights, 2024; 10(9), 1221-1236).

Ex vivo Gene Therapies:

Ex vivo genome editing (GE) cell and gene therapy (CGTs) are the most advanced in development (i.e., CASGEVY commercial approval) and comprise the majority of GE CGTs in the clinical. In most cases, delivery is achieved via electroporation to the target cells ex vivo while in vivo GE CGTs utilize viral and nonviral delivery platforms. Ex vivo GE CGTs offer an advantage of greater control of editing, as the target cell population can be precisely selected, and analysis of any off-target edits can be performed prior to administration to the patient. (Christina Fuentes “Coming of age: an overview of the growing toolbox for gene editing and its use in CGT applications” Cell & Gene therapy insights, 2024; 10(9), 1221-1236).

Ex vivo gene therapies include adoptive cell strategies that enhance the anti-tumour activity of lymphocytes to target rare blood cancers such as Kymriah and genetically modified CD34+ hematopietic stem cells to treat non-cancer related RD such as Strimvelis, Casgevy and Lenmeldy.

HSCs are the target cell population:

HSCs are a good target cell population in gene therapy due to their ability to repopulate the patient’s body and remain in the system indefinitely. One approach involves extracting the cells from the patient, culturing them ex vivo, genetically modifying them, and re-infusing them into the patient. In the meantime, the patient can receive chemotherpay or a conditioning regimen to create space in the bone marrow for the genetically modified cells. (Charlotte Barker “Advancing ene therapies for beta-hemoglobinopathies with novel genome and epigenome editing tools” Cell & Geen Therapy Insights, 2024: 10(9), 1163-1171).

In vivo Gene Therapies:

In vivo gene therapy involves direct administration (either locally or systemically) of the vectorized therapeutic genetic material with the intention of directly transfecting or transducing cells in situ for therapeutic effect. Vector systems for in vivo gene therapy are selected based on their specific targeting capacity for the intended tissue and their safety profiles. Currently, recombinant AAV has attracted the most attention, with seven AAV based products arleady on the market, including ntoable therapies such as Luxturna (for Leber congenital amaurosis (LCA)) and Zolgensma (for spinal muscular atrophy (SMA)).  (Henckaerts, “What re the issues associated with developing gene therapies for rare disease and are the current development models working?” Cell & Gene Therapy Insights 2024: 10(5), 773-784)

 RNA Therapy

Therapeutic RNA refers to antisense oligonucleotides (ASOs), such as gapmers, which contain DNA nucleotides flanked by RNA, small interfering RNAs (siRNAs) or large RNAs, such as messenger RNA (mRNA). These RNA therapies act by targeting RNA or proteins, by encoding missing or defective proteins, or by mediating DNA or RNA editing. Irrespective of their therapeutic mechanism of action, the large size of some therapeutic RNAs, such as mRNAs, their anionic charge, and their susceptibility to RNases present in both the bloodstream and tissues make it difficult for therapeutic RNA to enter cells efficiently and function on its own. To overcome the barriers to safe and effective RNA delivery, scientists have developed both viral vector and non-viral delivery systems that protect the RNA from degradation, maximize delivery to on-target cells and minimize exposure to off target cells. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022).

Oligonucleotide drugs, such as ASOs and siRNAs, that utilize enzymes endogenous to eukaryotic cells, such as RNAse H1 or the RNA induced silencing complex (RISC), respectively, facilitate delivery by not requiring the delivery of large enzymes. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022).

Although different RNA payloads can have different biochemical mechanisms of action, all of them must avoid clearance by off-target organs, must access the correct tissue, must interact with the desired cell type in a complex tissue microenviorment, must be taken up by endocytosis, and must exit the endosome, without eliciting a deleterious immune response. Although small oligonucleotide RNA therapeutics, including ASOs, siRNAs, and ADAR oligonucleotides, can be modified using stable chemistries and delivered using conjugates, mRNA based and DNA based therapeutics require a vehicle for entry into the cell. To facilitate this process, scientifists have developed several RNA delivery systems using a range of materials, including polymers and LNPs. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)).

RNA Interference (RNAi): (See outline)

Companies: Alnylam Pharmaceuticals

RNAi is emerging as a strategy for treating a range of genetic disorders. Its therapeutic efficacy is based on gene silencing wherein a synthetic siRNA takes advantage of machinery in the cell to inhibit specific mRNA expression. Unlike genome editing which make changes to the cell’s DNA, the effect of RNAi is not permanent, providing an extra layer of safety in the clinic. The approach has been used to treat liver fibrosis where siRNA loaded lipid nanoparticles are used to deactivate hepatic stellate cells (HSCs). The siRNA maket is estimated to surpass 67 billion by 2036. (Nnenna Ohaka “Aptamer shares jump 23% following siRNA deal with AstraZeneca”, Tides Global, July 16, 2024).

siRNA interferes with mature mRNA, which is easier to acheive than nuclear delivery. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)

mRNA:

Another type of RNA therapeutic is mRNA, which can encode proteins that have therapeutic activity. Because of their size, mRNAs are in vitro transcribed and cannot currently be made with site specific chemical modifications using solid state synthesis. mRNA can be used to replace protein, using replacement therapies, to reduct protein levels, using Cas9 cutting approaches, or to repair protein mutations at the DNA level, using base editing. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)).

Rare Diseases and Gene Therapy:

RD is an umbrella term used to group a road range of individual diseases that share the common trait of having a low point prevalence within a population. There are an estimated 7k forms of RD. One of the key therapeutic modalities opted for the development of new treatments for RD is gene therapy. Despite the unmet medical needs and clear rationale for gene therapy in a great many different forms of RD, the number of gene therapy products available in the clinic remains relatively low. From a specific in vivo gene therapy perspective, hundreds of clinical trails utilizing AAV based gene therapy strategies have been undertaken for a variety of indications, yet the number of in vivo gene therapies on the market is still in single digits. The case for ex vivo gene therapies is arguably better, but the nature of these medicines often limits them to rare, oncology and immunodeficiency related indications, restricting the number of RD indications with which they are applicable. Currently, as little as 5% of all RDs have pharmacological intervention options, leaving the remaining 95% with no access to established drug based treatments. One of the reasons for this disparity can be attributed to the complex commercialization models associated with developing pharmaceutical agents that target small patient populations. another problem associated with RD drug development is the lack of characterization within the diseases themselves. Of the 7k odd RDs, only 355 of them have a code in the existing International Classifcation of Diseases (ICD).  (Henckaerts, “What are the issues associated with developing gene therapies for rare disease and are the current development models working?” Cell & Gene Therapy Insights 2024: 10(5), 773-784))

Ophan Drug Act (OdA): was created in an attempt to provide incentivization for the development of new drugs for RDs. The act deployed tax incentives, enhanced patent protection and marketing rights, and clinical research subsidies to spur the pharmaceutical industry into action.

Adenoassociated viruses (AAVs):

Companies:  MeiraGTx (optimization of AAV vectors and promoter sequences. Riboswitch technology that is designed for control of gene expression by oral small molecules).

3PBIOVIAN

Introduction:

AAVs is one of the most commonly used viral vectors for in vivo gene therapies. However, AAV based treatments often cause a short-term heptic immune response. This can be dampened with the use of antinflammatory medications such as steroids. A number of methods have been prosed to prevent immune detection of gene therapies, including the use of lipid nanoparticles for gene delivery or engineering viral capsids to evade recognition for patients’ antibodies. (Rsellini, BioProcess International, vlume 20, number 4, april 2022.

An AAV’s protein shell surrounds and protects its small (25 nm) single stranded DNA genome of about 4.7 Kb. That genome contains just three genes; rep (for replication), cap (for the capsid) and nap 9 for particle assembly). Lacking viral DNA, recombinant AAV (rAAV) is essentially a protein based nanoparticle engineered to pass through cell membranes, through which it carries and delivers a therapeutic DNA “cargo” into the cell’s nucleus. Because AAVs cannot replicate without outside help, they provide a safe vehicle to drive long term transgene expression after single infection. (BioProcess international, 21(1-2) 2023).

Applications:

–-Duchenne Muscular Dystrophy (DMD):

DMD is a rare genetic muscle disease. It occurs primarily in males, although females can be affected and they also pass the genetic mutation onto their children. DMD affects an estimated 12,000 to 15,000 people in the U.S. and 25,000 in Europe. DMD is the most common childhood-onset form of muscular dystrophy. Loss of strength and function typically first appear between 3 and 5 years of age. DMD results from a genetic mutation in the DMD gene on the X chromosome. This gene regulates the production of dystrophin, a protein essential to healthy muscle development and function. In people with DMD, dystrophin levels are absent or nearly absent, which causes permanent damage to muscle cells. See Dyne

—-Elevidy (delandistrogen moxeparvovec rokl): is an adeno-assocaited virus vector based gene theurapy of ambulatory pediatric patients age 4-12 with Duchenne muscular dystrophy with a confirmed mutation in the Duchenne muscular dystrophy gene. It is designed to deliver into the body a gene that leads to production of Elevidys micro-dystrophin that contains selected domains of the dystrophin protein present in normal muscle cells. The accelerated US Food and Drug Administration (FDA) approval of delandistrogene moxeparvovec was based on data from a randomized clinical trial that established that delandistrogene moxeparvovec increased the expression of the Elevidys micro-dystrophin protein observed in delandistrogene moxeparvovec-treated individuals aged four to five years with Duchenne muscular dystrophy.

–Spinal Muscular Atrophy:

SMA is a neurological disorder caused by a mutation in the SMN1 gene, which leads to a decrease in the SMN protein, a protein encessary for the survival of motor neurons.

—-Zogensma (Onasemnogene abeparvovec) (Novartis): is a gene therpay used to treat pediatric patients less than two years of age with spinal muscular atrophy with biollelic mutations in the survival motor nueron 1 gene. It involves a 1 time infusion of the drug into a vein. Zogensma is a biological drug which uses AAV9 viral capsids containing an SMN1 transgene.

–-REE65 Mediated Retinal Dystrophy:

Lentiviral Gene Therapies

Companies:  Avrobio  (AVRO: develops gene therapies to treat lysosomal storage diseases)

Lentiviral vecors are used mosly for ex vivo gene therpapy and are designed to deliver stable and durable integration of transgenes into a patient’s hematopoietic stem cells. The prcoess begins with harvesting CD34 stem cells form a patient. These cells are modified by using a lentiviral vector to insert a transgene that will enable production of a functional protein. The modified stem cells are infused back into the patient, where they engraft in the patient’s bond marrow. The cells divide to produce daughter cells, all carrying the transgene for the functional protein needed by those cells. In some cases, a protein is secreted to help other cells that have the defective gene, a process called cross-correction.

–Luxturna (voretigene neparvovec-rzyl): is an deno-assocaited virus vector based gene therpay approved for patients with confirmed biallelic RPE65 mutation-assocaited retinal dystrophy. Patients must have viable retinal cells as determiend by the treating physician(s). The gene therapy is not a cure for the condition, but substantially improves vision in those treated.

Non-viral vectors for gene delivery have attracted attention in the past decades becasue of their potential for limited immunogenicity, the ability to accomodate and deliver alrge size genetic materials and the potential for modification of theri surface structures.

Noevertheless, inherent to nonviral vectors are also important challenges. First, nonviral systems face aprpeciable plasmid loss during their mitotic segration in dividing cells. Second, silencing of transgene expression may occur due to epigentic events. Their, there is variable efficiency of plasmid delivery into various cells, both by physical and chemical methods. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

The major categories of non-viral vectors include cationic lipids and cationic polymers. 

Cationic lipid derived vectors:

Cationic lipid dervied vectors represent the most extensively investigated systems for non-viral gene delviery. Cationic polymer non-viral vectors have gained increasing attention becasue of flexibility in their synthesis and structural modifictions for specific biomedical applications. Both cationic lipid and cationic polymer systems deliver genes by forming condensed complxes with negatively charge deoxyribonucleic acid (DNA) through electrostatic interactions. Polyplexes formed between cationic polyemrs and DNA are relatively more stable than lipoplexes formed between cationic lipds and DNA. (Zhou “Biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery” Nat Mater: 11(1) 82-90 (2012). 

Lipids and Lipid-based nanoparticles:

Companies: Beam Therapeutics

Polymers, liposomes, or other nanoscale structures can be modified to enhance their biocompatility, reduce cytotoxicity (e.g., PEG), and augment their potential to target specific tisseus or cell types. Polyemr and liposme ehcists increasingly collaborate with biologists to optimize the capablity of syntehtic nanostructures for improving nonviral gene delivery. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Concurrent advances in the development of syntehtic materials that encapsulate RNA, such as polymers, lipids and lipid nanoparticles (LNPs) ahve invigorated research into non-viral based delivery systems leading to FDA approval of subutaneously administered N-acetylgalactosamine (GalNAc)-siRNA conjugates that target hepatocytes, intravenously adminsitered LNP based siRNA drugs that target hepatocytes and emergency use authorizaton (EUA) and FDA approval for intramuscularly administered LNP based mRNA COVID vaccines. Tehse approvals suggest that improved delivery to non-liver tissues (also known as extrahepatic tissues) as well as local deliveyr to the CNS, eye and ear oculd result in new drugs. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022).

LNPs:

Lipid Nanoparticles (LNPs) are a key class of drug delivery system that includes nanoparticles approved by the FDA for liver siRNA delivery and form fRNA vaccine delivery. On the basis of the size of the hydrophoilic head group relative to the size of the hydrophobi tail or tails, lipids form distinct structures including micelles, liposomes and LNPs. FDA approved LNPs contain variations of four basic components: a cationic or ionizalbe lipid cholesterol, a helper lipid, and a poly (ethylene glycol) (PEG) lipid. In addition to the RNA payload, the Alnylam, Moderna and Pfizer/BioNTech/Acuitas LNPs include four components: the cationic or ionizable lipids DLin-MC3-DMA (Alnylam), SM-102 (Moerna) or ALC-0315 (Pfizer/BioNTech/Acuitas), cholesterol, the PEG-lipids PEG-2000-C-DMG (Alnylam), PEG-2000-DMG (Moderna) or ALC-0159 (Pfizer/BioNTech/Acuitas) adn DSPC. (Dahlman, “Drug delivery systems for RNA therapeutics” Nature Reviews Genetics, 23, May 2022)

LNPs are a key class of drug delivery system that includes nanoparticles approved by teh FDA for liver siRNA delivery and for mRNA vaccine delivery. On the basis of the size of the hydrophilic head group relative to the size of the hydrophobic tail or tails, lipids form distinct structures including micelles, liposomes and LNPs. FDA aprpoved LNPs contain variations of four basis components: a cationic or ionizable lipid, cholesterol, a helper lipid and poly(ehtylene glycol) (PeG)-lipd. Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)

Moderna discloses lipids and compounds useful for the nanopartcile composition for delivery of RNA therapeutics. (see US 9,533,047, US 9,867,888, US 10,195,156, 10,933,127  and US 10,799,463).

In addition to the RNA payload, the Alnylam, Moderna and Pfizer/tioNTech/Acuias LNPs comprise four componetns: the cationic or ionizable lipdis DLin-MC3-DMA (Alnylam), SM-102 (Moderna) or ALC-0315 (Pfizer), cholesterol, the PEG-lipids PEG-2000-C-DMG (Alnylam), PEG-2000-DMG (Moderna) or ALC-0159 )Pfizer). Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)

Liposomes and synthetic polymers are chemical means that exploit the nanoscale size and cotrollable surface properties of organic and polyemric molecuels for gene delivery. These chemical carreirs are intended ot match or exceed the performance of viral vectors with fewer immunogenic complciaitons. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

–PEGylated siRNA-loaded lipid nanopartciles: have addressed the greatest challenge in implementing siRNA therapeutics, which is their delivery. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

–In vivo Gene therapies:

In vivo gene therapeis can be delivered via the nonviral vehicle lipid nanoparticle (LNP). In fact, gene editing tools can be delivered to patients through intravenous infusion of LNPs targeted to hematopietic stem cells (HSCs), elimianting the need for transplantation altogtther. 

Virus-Like Particles:

Virus-like particles represent another class of nonviral gene dleivery vectors, and these typically consist of self assembled viral protein nanostructures. 

Polymers and Polymer-based nanoparticles:

Many non-viral RNA delivery systems utilize polymers and polymeric nanoparticles. Chemists can vary polymer traits including charge, degradability and molecualr weight, all of which influence how polymers delivery RNA into cells. One frequently used polymer is poly(lactic-co-glycolic acid) (PLGA). PLGA drug delivery systems have been approved by the FDA for the delivery of small molecule drugs but not for the devliery of nucleic acids. At neutral pH, PLGA does not have the positive charge required to complex the anionic RNA phosphodiester backbone. Thus, to utilze PLGA as an RNA deliveyr system, scientists have added cationic chemical groups such as chitosan to delivery siRNA in mice. (Dahlman, “Drug delivery systems for RNA therapeutics” Nature Reviews Genetics, 23, May 2022)

Episomal vectors/Episomes/Exosomes/Plasmids: 

Most plasmids (natural or aritifical) used in trasnfections, remain in the nucleus only transiently. Nonviral episomes have to pass thorugh a number of critical stages, starting with gaining cellular entry, until they can modulate sustained, long term maintenance within cells. The fate of pEPI-based, established, and onintegrating episomal vectors is characterized by a sequence of fundamental stages inside the host cell: establishment, replication, mitotic stability, and plasmid segregation in daughter cells at mitosis. These are ocmplex processes mediated by specific chromatin structures and DNA features, and studies during the recent years have greatly explanded how episomes can function as gene transfer vectors in a gene therapy context. Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Exosomes:

Exosomes are small secreted vesicles that are produced by all cells, are present at high concentrations in interstitial and other body fluids, and have been present throughout the life of every person and the evolution of every animal. They are in fact the only biologically nomral nanovesicle in existence. In contrast to LNPs, which have been shown to elicit cellular toxicity, exosomes were shown to have no adverse effects at any dose tested. Moreover, mRNA-loaded exomsomes were characterize by efficient mRNA encapsulation, high mRNA content, consistent and consistent size. (Gould, “Exosome-medaited mRNA delivery in vivo is safe adn can be used to induce SARS-CoV-2 immunity” J. Biiological Chemistry, 2021). 

Nonviral and nonintegrating episomal vectors are reemerging as a valid, alternative technology to integrating viral vectors for gene therapy, due to their more favorable saftey profile, significantly lower risk for insertional mutagenesis, and a lesser potential for innate immune reacitons, in addition to their low production cost. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Selaru (WO 2016/123556) discloses extracellular vesicles (Evs) derived from a cancer assocaited fibroblast (CAF) which includes a payload such as a polynucleotide such as miR-195, miR-126 or miR-192, identified as being dwon-regulated in the CAF. The extracellular vesicel selectively target a cancer cell. 

Episomes:

The term episome was propsoed by Fancois Jacob and Elie Wollman in 1958 to describe extrachromosomal genetic material that may replicate autonomously or become integrated into the chromosome. Although the term episome is now somewhat interchangeable with the term plasmid, episomes are larger in size and are retained for a longer period of tiem following transfection of host cells. Episome integration is a rare event and can occur through canonical sequence independent, nonhomolgous end joining or microhomology medaited end joining. Nevertheless. episomal integration must be inhibited if episomes are to become safe vihciles for gene therapy applciations. Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Episomal vectors or “episomes” are free, circular, extrachromosomal DNA molecuels of viral and nonviral origin. Viral epiosmes refer to viruses whose natural life cycle includes a stage of remaining as free, viral DNA within the cell nucleus, retaining the ability to encode proteins without integrating into the host cell’s genome. Examples include Adenoviruses (see outline). Non-viral episomes are effectively plasmids, usually of a larger size relative to conventional plasmids, and can exist independent of the genomic DNA for longer periods than covnentional plasmids. Both types of episomes are used as vectors of gene transfer in gene therpay. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Successful episome based gene expression hinges on the ability to safely and precisely deliver episome vectors to complex biological environments. Although bare nucleic acids can be delivered in vivo by direct introduction of DNA or RNA into cells, rpaid clearance and loss of expression limit the effectiveness of this approach. Several carreir systems can be utilized to deliver episomes and these include liposomes, syntetic polyemrs, or physical means of gene delivery (electroporation and sonoporation, among others). (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Episomes have also been successfully delivered in several tissues using lysin-PEG and polycationic comb polymers with nuclear loalizing sequences, among others. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

–Types of episomal vectors:

The most commonly used episomal vector ahs been pEPI-1, which originated form pGFP-C1 with the addition of the S/MARs anchoring elements, which keep the chromosomal DNA tethered into the nuclear matrix or scaffold. pEPI-1 was the first vecotr reproted as nonintegrative in the long term following trasnfection and the maintenance of key vecotr egiones. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Sleeping Beauty (SB) Transporson System:

The sleeping Beauty (SB) transposon system has emerged as a promising alternative to viral vectors. The SB system shares with viral vectors the need to integrate into the recipient cell’s genome to be functional. However, ocntrary to viral vectors, it does not show integration preference for sites within highly transcribed gehome domains. And since the vast majority of the genome does not contain highly transcribed gehnome domains, random integraiton of SB outside these domains reduces its risk for insertional mutagenesis. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

–Modified mRNA: Modern disclsoes a modified mRNA which overcome probleems with respect to the modulation of intracellular translation and processing of nuceic acids encoding polypetpides (US 9,950,068).